Bilirubin is a degradation product of hemoglobin. According to estimates, approximately 6 to 7 grams of hemoglobin are released from damaged or aged red blood cells each day. The liver, spleen, and bone marrow rapidly degrade this pool of hemoglobin resulting in approximately 200 to 230 milligrams of bilirubin and its derivatives being formed each day in the normal human adult. Subsequently, as a part of normal human metabolic processes the major portion of this daily bilirubin production is excreted, degraded into other derivatives, etc.
In some cases, however, an excessive amount of bilirubin occurs within the human body through overproduction of bilirubin, as in the case of excessive hemolysis, or by retention of bilirubin due, for example, to a liver failure. Invariably, jaundice results. This widely encompassing pathological condition is characterized by markedly elevated serum bilirubin levels, for example, 10 milligrams of bilirubin per deciliter of serum or higher compared to the normal adult range of 0.1 to about 1 milligram of bilirubin per deciliter of serum. There is generally also present a brownish-yellow pigmentation of the skin, sclera, or mucous membranes.
In addition, increasing evidence suggests that excess amounts of bilirubin in the blood can lead to an undesirable increase in bilirubin concentration within body cells and interfere with various cellular processes. For example, bilirubin has been widely implicated as a potent inhibitor of many enzymatic reactions that generate energy vital to the cell. Given this background, the clinical diagnostic significance of bilirubin in tests for liver and other related organ functions is self-evident.
Perhaps the most widely used analytical procedure for bilirubin assay work has been the so-called diazo method. The diazo method employs a coupling reaction of bilirubin with a diazonium salt, such as diazosulfanilic acid, to form a pigment having an extinction coefficient higher than bilirubin by itself (which has a yellowish coloration). The diazo method, however, has a variety of problems. For example, as noted in Clinical Chemistry-Principles and Technics, edited by R. J. Henry, D. C. Cannon, and J. W. Winkelman, Harper and Row Publishers, 2nd Edition, pages 1042-1079 (1974), because of the many variants and the complexity of the diazo method, the determination of bilirubin for a given sample is often quite different for different variants of the diazo method. In addition, the diazo method can be time-consuming because it typically requires several reagents which generally are freshly mixed for each determination. Moreover, the diazo method can be inaccurate because certain body fluid components other than bilirubin respond to diazotization.
Wu et al, U.S. Pat. No. 4,069,017 issued Jan. 17, 1978, describes a new assay for the determination of bilirubin. This patent discloses that certain interactive mordant compositions can be effectively employed to mordant bilirubin. Such mordanted bilirubin greatly facilitates the colorimetric detection of bilirubin in an aqueous liquid sample. The mordanted bilirubin exhibits a marked increase in molar extinction coefficient and a shift in absorption peak in comparison to that of free bilirubin.
The assay method of U.S. Pat. No. 4,069,017 is colorimetric, and there is no disclosure of modifying this method to provide a fluorimetric assay. Such modifications would be highly advantageous for at least several reasons. First, as is well-known, fluorimetric assays are capable of greater sensitivity than is possible with colorimetric assays. Second, although the assay method described in U.S. Pat. No. 4,069,017 is colorimetric, it has many advantages which would also be useful in a fluorimetric assay. For example, the assay method of U.S. Pat. No. 4,069,017 is direct, quantitative, substantially free of interferents, and can be performed with ease.
Based on the foregoing reasons, any adaptations or modifications of the colorimetric bilirubin assay described in U.S. Pat. No. 4,069,017 to provide a fluorimetric bilirubin assay would clearly represent an extremely valuable contribution to the art.